Surface modified electrodes, and methods of preparation thereof

ABSTRACT

A surface modified electrode is provided. The surface modified electrode includes a glassy carbon electrode (GCE) and a nanomaterial disposed on the glassy carbon electrode. The nanomaterial comprises carbon nanotubes (CNTs), and at least one of thallium oxide nanoparticles (Tl 2 O 3 ·NPs), thallium oxide (Tl 2 O 3 ) nanopowder, and thallium oxide carbon nanotube nanocomposites (Tl 2 O 3 ·CNT NCs). A polymer matrix is configured to bind the glassy carbon electrode with the nanomaterial. A method of preparing the surface modified electrode is also disclosed. The surface modified electrode can be implemented in a biosensor for detecting a biological molecule, like choline.

TECHNICAL FIELD

The present disclosure relates to a surface modified electrode, and morespecifically, the present disclosure relates to the surface modifiedelectrode for use in a biosensor for detecting a biological molecule.

BACKGROUND

Choline (2-hydroxy-N,N,N-trimethylethanaminium) is a water-solubleammonium molecule required for different biological functions. It is animportant predecessor of the acetylcholine neurotransmitter that isconcerned for muscle and memory control. Few important phospholipids,such as phosphatidylcholine, that are responsible for trans-membranesignaling and the structure of the cell membrane, can be synthesizedfrom choline. Primary diagnosis of brain disorders, such as, parkinson'sand alzheimer's disease can be analyzed clinically through quantitativeassessment of choline. Increased level of choline has been reported tocause an enhanced threat of cancer and DNA damage. Hence, monitoring thecholine level is critical in many cases.

Conventional methods for determination of choline, likechemiluminescence, fluorescence, and proton nuclear magnetic resonancesuffer from drawbacks such as complexity in operation, long detectiontimes, and high cost. Electrochemical sensors based on enzymaticapproach is one of the progressive techniques for the detection ofcholine in natural science areas such as environmental, industrial, andclinical aspects since it offers rapid, cheap, and simple operation butits dependency on the enzymatic environment makes the applicationrestricted. Owing to such drawbacks, there exists a need to provide asimple, cost-effective, reliable, method for non-enzymatic detection ofcholine with a short response time.

SUMMARY

The present disclosure relates to a surface modified electrode. Thesurface modified electrode can be implemented in a biosensor fornon-enzymatic detection of biomolecules, particularly choline. Thepresent disclosure also relates to a method of preparing the surfacemodified electrode.

In one aspect of the present disclosure, the surface modified electrodeis disclosed. The surface modified electrode includes a glassy carbonelectrode (GCE) and a nanomaterial disposed on the glassy carbonelectrode. The nanomaterial includes carbon nanotubes (CNTs), and atleast one of thallium oxide nanoparticles (Tl₂O₃·NPs), thallium oxide(Tl₂O₃) nanopowder and thallium oxide carbon nanotube nanocomposites(Tl₂O₃·CNT NCs). In certain embodiments, the carbon nanotube is asingle-walled carbon nanotube (SWCNT), a double-walled carbon nanotube(DWCNT), a multi-walled carbon nanotube (MWCNT), or any combinationthereof. The surface modified electrode further includes a polymermatrix configured to bind the glassy carbon electrode with thenanomaterial. In some embodiments, the polymer matrix is a sulfonatedtetrafluoroethylene-based fluoropolymer (nafion).

The surface modified electrode can be implemented in a biosensor fordetecting a biological molecule. In some embodiments, the biologicalmolecule is one selected from a group consisting of acetylcholine,ascorbic acid, cholesterol, choline, dopamine, folic acid, L-glutamicacid, L-glutathione, L-tyrosine, and uric acid. In some embodiments, thebiological molecule is choline. In an embodiments, the biosensor isconfigured to detect choline across a concentration range of 100.0 pM to100.0 mM. In some embodiments, the biosensor is configured to detectcholine having stability for about 30 days. In certain embodiments, thebiosensor is configured to detect choline with a detection limit as 9.14pM, a sensitivity of 104.68 μAμM⁻¹ cm⁻², a linear dynamic range in therange of 100.0 pM-1.0 mM, and a linearity value in the linear dynamicrange as 0.9884.

In another aspect of the present disclosure, a method of preparing thesurface modified electrode is described. The method includes mixingcarbon nanotubes (CNT's), and at least one of thallium oxidenanoparticles (Tl₂O₃·NPs), thallium oxide (Tl₂O₃) nanopowder, andthallium oxide carbon nanotube nanocomposites (Tl₂O₃·CNT NCs), in anorganic solvent to form a slurry of the nanomaterial. The method alsoincludes disposing the slurry of the nanomaterial on the glassy carbonelectrode to form a film. In certain embodiments, the slurry of thenanomaterial is disposed on the glassy carbon electrode for a period of1-3 hours at a temperature range of 35-40° C. Further, a polymer matrixis coated on the film to obtain the surface modified electrode. In someembodiments, the polymer matrix is coated on the film for a period of2-4 hours at a temperature range of 35-40° C. The polymer matrix is asulfonated tetrafluoroethylene-based fluoropolymer (nafion).

In some embodiments, the method further includes obtaining the thalliumoxide carbon nanotube nanocomposites (Tl₂O₃·CNT NCs) by stirring a saltof thallium in water in an alkaline medium to form a first mixture. Insome embodiments, the salt of thallium is stirred in water in analkaline medium for a period of 5-7 hours at a temperature range of 80°C.-100° C. In an example, the salt of thallium is thallium nitrateThNO₃)₃. The method further includes washing the first mixture to obtaina second mixture, and drying the second mixture to obtain the thalliumoxide carbon nanotube nanocomposites. In certain embodiments, the secondmixture was dried for a period of 10-14 hours at a temperature range of25-37° C.

The foregoing as well as other features and advantages of the presentdisclosure will be more fully understood from the following description,examples, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of this disclosure and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

FIG. 1 is an exemplary flowchart illustrating a method for preparing thesurface modified electrode;

FIG. 2A shows a ultraviolet (UV) spectrum of thallium oxide (Tl₂O₃)nanoparticles;

FIG. 2B is a bandgap energy plot of Tl₂O₃ NPs;

FIG. 2C shows the UV spectrum of thallium oxide carbon nanotubenanocomposites (Tl₂O₃·CNT NCs);

FIG. 2D is the bandgap energy plot of the Tl₂O₃·CNT NCs;

FIG. 3A shows a fourier-transfer infrared spectroscopy (FT-IR) spectrumof carbon nanotubes (CNT), thallium oxide (Tl₂O₃) nanoparticles, andthallium oxide carbon nanotube nanocomposites (Tl₂O₃·CNT NCs);

FIG. 3B shows X-ray diffraction (XRD) studies of CNTs, Tl₂O₃nanoparticles, and Tl₂O₃·CNT NCs;

FIG. 4A shows Field Emission Scanning Electron Microscope (FESEM) imageof CNT;

FIG. 4B shows FESEM image of Tl₂O₃ NPs;

FIG. 4C shows FESEM image of Tl₂O₃·CNT NCs;

FIG. 4D shows elemental analysis of Tl₂O₃·CNT NCs;

FIG. 5A shows comparative binding energy examination of CNT, Tl₂O₃ NPs,Tl₂O₃·CNT NCs;

FIG. 5B shows binding energy examination of Tl³⁺;

FIG. 5C shows binding energy examination of C1s;

FIG. 5D shows binding energy examination of O1s;

FIG. 6 is a flowchart depicting a method for fabrication of Tl₂O₃·CNTNCs on glassy carbon electrode with a polymer matrix (nafion), aproposed electrochemical mechanism of choline at surface modifiedelectrode of the present disclosure, and also depicts outcomes of I-Vexperimental results;

FIG. 7A is a voltammogram depicting the effect of pH on the surfacemodified electrode in the presence of choline within a pH range of5.7-8.0;

FIG. 7B is a voltammogram comparing the electrochemical behavior with abare electrode (glassy carbon electrode), glassy carbon electrode with apolymer matrix (nafion) and the surface modified electrode, in sensingcholine;

FIG. 7C illustrates a I-V graph depicting the selectivity of the surfacemodified electrode towards various biomolecules;

FIG. 7D is a voltammogram comparing the effect of choline on glassycarbon electrode, glassy carbon electrode with a polymer matrix (nafion)and the surface modified electrode;

FIG. 8A is an I-V graph depicting the effect of concentration of cholineon current change with the surface modified electrode;

FIG. 8B shows a calibration plot obtained from FIG. 8A vs choline;

FIG. 8C shows a linear dynamic range plot with an error limit of 10%;

FIG. 8D shows a response time of choline towards the biosensor;

FIG. 9A is a plot comparing the effect of current change with theTl₂O₃·CNT NCs coated electrode sensor with that of bare electrode sensorin responses to choline;

FIG. 9B shows bar diagram depicting the effect of current change withthe Tl₂O₃·CNT NCs coated electrode sensor with that of bare electrodesensor in response to choline at +1.2 V with error bar 10.0%;

FIG. 10A shows the reproducibility response of Tl₂O₃·CNT NCs coatedelectrode for choline sensing;

FIG. 10B shows the repeatability response of Tl₂O₃·CNT NCs coatedelectrode for choline sensing;

FIG. 11A is a plot depicting interference analysis of the Tl₂O₃·CNT NCscoated electrode in the presence of other interfering biomolecules;

FIG. 11B shows a bar diagram depicting current change response to theinterfering biomolecules at +1.3 V with an error limit of 10.0%; and

FIG. 12 is an I-V graph determining choline concentration from variousbiological samples with the sensor of the present disclosure

DETAILED DESCRIPTION

Reference will now be made in detail to specific embodiments orfeatures, examples of which are illustrated in the accompanyingdrawings. Wherever possible, corresponding or similar reference numberswill be used throughout the drawings to refer to the same orcorresponding parts. Moreover, references to various elements describedherein, are made collectively or individually when there may be morethan one element of the same type. However, such references are merelyexemplary in nature. It may be noted that any reference to elements inthe singular may also be construed to relate to the plural andvice-versa without limiting the scope of the disclosure to the exactnumber or type of such elements. A skilled artisan will appreciate thatvarious alternate embodiments and forms may be prepared. Examples,therefore, given are only for illustration purposes without anyintention to restrict the embodiments to a given set of examples.Specific functional aspects are provided merely to enable a personskilled in the art to perform the invention and should not be construedas limitations of the invention. Any method steps and processesdescribed herein are not to be construed as necessarily requiring theirperformance in the particular order discussed or illustrated, unlessspecifically identified as an order of performance. It is also to beunderstood that additional or alternative steps may be employed, unlessotherwise indicated.

As used herein, “surface modified electrode” refers to an electrodetreated to modify the surface properties, and enhance electrochemicalfunctions.

As used herein, “nanomaterial” refers to chemical substances ormaterials having particle sizes between 1 to 100 nanometers in at leastone dimension.

As used herein, “carbon nanotubes (CNTs)” refers to a single-walledcarbon nanotube (SWCNT), a double-walled carbon nanotube (DWCNT), amulti-walled carbon nanotube (MWCNT).

The term “glassy carbon” refers herein to a non-graphitizing carbonwhich combines glassy and ceramic properties with those of graphite.

As used herein, “mixing” refers to combining two or more materialstogether with the use of a mixer or any other device.

As used herein, “working electrode” refers to the electrode in anelectrochemical cell/device/biosensor on which the electrochemicalreaction of interest is occurring.

As used herein, “counter-electrode”, is an electrode used in anelectrochemical cell for voltametric analysis or other reactions inwhich an electric current is expected to flow.

As used herein, “limit of detection (LOD)” is the smallest concentrationof an analyte in a test sample that can be easily distinguished fromzero.

As used herein, “limit of quantification (LoQ)” is the smallestconcentration of an analyte in the test sample that can be determinedwith acceptable repeatability and accuracy.

As used herein, “linear dynamic range (LDR)” is the range ofconcentrations where the signals are directly proportional to theconcentration of the analyte in the sample.

As used herein, “selectivity” is the quality of the electrochemicalresponse that can be achieved without interference for any othersubstance.

As used herein, “sensitivity” is the change in the electrochemicalresponse with regard to a change in the concentration of the analyte.

As used herein, a “voltammogram” is a graph that can be drawn after anelectrochemical experiment. This graph has a typical, recognizable formin which the electron flow (current: I) is measured in Volt against thepotential (E).

As used herein, “amount” refers to the level or concentration of one ormore elements or end-products of the system and the methods of thepresent disclosure.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having,” “comprise,” “comprises,” “comprising” or the like should begenerally understood as open-ended and non-limiting unless specificallystated otherwise.

It is understood that the order of steps or order for performing certainactions can be changed so long as the intended result is obtained.Moreover, two or more steps or actions may be conducted simultaneously.

As used herein, the term “about” or “between” refers to a ±20% to ±10%variation from the nominal value unless otherwise indicated.

Embodiments of the present disclosure are directed towards a surfacemodified electrode. The surface modified electrode consists of a glassycarbon electrode (GCE) modified/fabricated with carbon nanotube thalliumoxide based nanomaterial. The surface modified electrode whenimplemented in a biosensor functions as a working electrode, and iseffective in detection of biomolecules with high selectivity,sensitivity, across a wide concentration range, with a short responsetime. In an example, the biomolecule is choline. The electrochemicalcharacteristics of the surface modified electrode were found to be muchsuperior in comparison to the bare electrode or the glassy carbonelectrode, confirming the superiority of the carbon nanotube thalliumoxide based nanomaterial disposed on the biosensor. Although, thepresent disclosure describes the use of the biosensor for detection ofcholine in a non-enzymatic environment, the sensor of the presentdisclosure may be adapted for detection of other biomolecules as well.

In an embodiment, the surface modified electrode includes a glassycarbon electrode modified/fabricated with a nanomaterial disposed on atleast a portion of the glassy carbon electrode. In an example, thenanomaterial may be disposed across the length of the glassy carbonelectrode with a uniform thickness or may be diposed on portions of theglassy carbon electrode. The fabrication of the nanomaterial over theglassy carbon electrode may be done by any conventional methods known inthe art. In an example, the fabrication is done by drop castingtechnique. The nanomaterial includes carbon nanotubes (CNT's) inaddition to one or more of thallium oxide nanoparticles (Tl₂O₃·NPs),thallium oxide (Tl₂O₃) nanopowder and thallium oxide carbon nanotubenanocomposites (Tl₂O₃·CNT NCs). In an embodiment, the glassy carbonelectrode is disposed with CNT's, Tl₂O₃·NPs, Tl₂O₃ nanopowders, andTl₂O₃·CNT NCs. In another embodiment, the glassy carbon electrode isdisposed with CNT's, Tl₂O₃·NPs and Tl₂O₃·CNT NCs. The carbon nanotubemay be a single-walled carbon nanotube (SWCNT), a double-walled carbonnanotube (DWCNT), a multi-walled carbon nanotube (MWCNT), or anycombination thereof.

The surface modification of the glassy carbon electrode with the carbonnanotubes and the nanomaterial is to impart desirable physical andchemical properties, such as increased surface area, or adsorption orabsorption capacity to biomolecules of interest. In an example,thickness of the nanomaterial diposed on the glassy carbon electrode isabout 10 nanometer to about 50 nanometer. The surface modified electrodefurther includes a polymer matrix that is configured to bind the glassycarbon electrode with the nanomaterial by a chemical bond. In anexample, the nature of bonding between the nanomaterial and the glassycarbon electrode, facilitated through the polymer matrix, is a covalentbond. In another example, the nature of bonding between the nanomaterialand the glassy carbon electrode is physical adsorption. In someembodiments, the polymer matrix may be a sulfonatedtetrafluoroethylene-based fluoropolymer (nafion or NFN). The surfacemodified electrode is GCE/Tl₂O₃·CNT NCs/NFN

The present disclosure also describes a biosensor for detecting thebiological molecule. The biosensor is a two-electrode system having thesurface modified electrode as a working electrode, and a platinum wireas a counter-electrode. The working electrode and the counter-electrodemay be connected to each other by way of electrical interconnects thatallow for passage of current between the electrodes, when a potential isapplied between them. The working electrode and the counter-electrodemay be arranged as obvious to a person of ordinary skill in the art. Inan example, the electrode configuration of the electrochemical sensormay be designed based on the type of the biomolecule to be detected andtype of detection methodology. Although the present disclosure describesa two-electrode system (the working electrode and the counter-electrode)in the sensor, the sensor may be adapted to have a 3-electrode or a4-electrode or a multi-electrode system to detect one or morebiomolecules of interest. In certain embodiments, the working electrodehas a cross-section diameter of 1.68 millimeters, and thecounter-electrode as a cross-section diameter of 0.2 millimeters. Incertain other embodiments, the working electrode and thecounter-electrode can have same dimensions.

The biosensor having the surface modified electrode can be used todetect one or more biological molecules. In certain embodiments, thebiological molecule may be one selected from a group consisting ofacetylcholine, ascorbic acid, cholesterol, choline, dopamine, folicacid, L-glutamic acid, L-glutathione, L-tyrosine, and uric acid. In oneembodiment, the biological molecule is choline. Although embodiments ofthe present disclosure are directed towards detection of choline, it maybe understood by a person of ordinary skill in the art that thebiosensor may be adapted for detection of other biological molecules aswell.

The sensor becomes operable when the biomolecule of interest, such ascholine is brought in contact with the working electrode. A chemicalreaction between the working electrode and the biological moleculeoccurs causing a change in chemical information associated with thebiological molecule. In an example, the change in chemical informationcould be a change in oxidation state. In other words, the biologicalmolecule may undergo a redox (oxidation-reduction) reaction resulting inloss of electrons, when it is brought in contact with the workingelectrode. The sensor is configured to determine a change in chemicalinformation caused by the biological molecule on contact with at least aportion of the surface modified electrode, and further transduce thechange in chemical information associated with the biological moleculeto an electrical signal. In certain embodiments, the electrical signalis indicative of a concentration level of the biological molecule.Therefore, the greater the concentration of the biological molecule, thestronger is the signal.

In some embodiments, the biosensor may be configured to detect cholineacross a concentration range of 100.0 pM˜100.0 mM.

In another embodiment, the biosensor may be configured to detect cholinehaving stability for up to about 30 days.

In yet another embodiment, the biosensor configured to detect cholinehas a detection limit of sensor as 9.14 pM.

In another embodiment, the biosensor configured to detect choline has asensitivity of 104.68 μAμM⁻¹ cm⁻².

In another embodiment, the biosensor configured to detect choline has athe linear dynamic range as 100.0 pM-1.0 mM.

In yet another embodiment, the biosensor configured to detect cholinehas a linearity value in the linear dynamic range as 0.9884.

Referring to FIG. 1A, a method for preparing the surface modifiedelectrode is described. In an embodiment, the method 100 includes mixingcarbon nanotubes (CNT's), and at least one of thallium oxidenanoparticles (Tl₂O₃·NPs), thallium oxide (Tl₂O₃) nanopowders, andthallium oxide carbon nanotube nanocomposites (Tl₂O₃·CNT NCs), in anorganic solvent to form a slurry of the nanomaterial (102). In anembodiment, the CNT's, Tl₂O₃·NPs, Tl₂O₃ nanopowders, and Tl₂O₃·CNT NCsare mixed in an organic solvent to form a slurry of the nanomaterial. Inanother embodiment, the CNT's, Tl₂O₃·NPs and Tl₂O₃·CNT NCs are mixed inan organic solvent to form a slurry of the nanomaterial. The carbonnanotube may be a single-walled carbon nanotube (SWCNT), a double-walledcarbon nanotube (DWCNT), a multi-walled carbon nanotube (MWCNT), or anycombination thereof. The organic solvent is a lower alcohol, such asethanol. The method further includes diposing the slurry of thenanomaterial on the glassy carbon electrode to form a film. In certainembodiments, the method includes disposing the slurry of thenanomaterial with the glassy carbon electrode for a period of 1-3 hoursat a temperature range of 35-40° C. (104).

The method also includes coating the polymer matrix on the film toobtain the surface modified electrode (106). In one embodiment, themethod includes coating the polymer matrix on the film for a period of2-4 hours at a temperature range of 35-40° C. In another embodiment, thepolymer matrix is a sulfonated tetrafluoroethylene-based fluoropolymer(Nafion). In certain embodiments, the polymer matrix may be added to theglassy carbon electrode in a drop-wise manner and kept open in the airso as to synchronize coating development.

In some embodiments, a method for preparing the thallium oxidenanocomposites (Tl₂O₃·CNT NCs) is described. The method includesstirring a salt of thallium in water in an alkaline medium to form afirst mixture. The salt of thallium may be thallium nitrate Tl(NO₃)₃. Inone embodiment, the method includes stirring the salt of thallium inwater in an alkaline medium for a period of 3-8 hours at a temperaturerange of about 70° C.-110° C. In another embodiment, the method includesstirring the salt of thallium in water in an alkaline medium for aperiod of 5-7 hours at a temperature range of about 80° C.-100° C. Inyet another embodiment, the method includes stirring the salt ofthallium in water in an alkaline medium for a period of 4 hours at 90°C. In certain embodiments, the alkaline medium of the first mixture maybe obtained by the addition of a base component such as, but not limitedto, NaOH, KOH, Ca(OH)₂, or a combination thereof. In some embodiments,the stirring may be done by equipment such as, but not limited to,magnetic stirrer, stirring motors, shakers, and small pumps, etc. Themethod further includes washing the first mixture to obtain a secondmixture. In certain embodiments, the first mixture may be washed withwater and acetone and kept in the open air for 1.5 hours at roomtemperature. The method also includes drying the second mixture toobtain the thallium oxide nanocomposites. In one embodiment, the methodmay include drying the second mixture for a period of 8-16 hours at atemperature range of about 22° C.-40° C. In another embodiment, themethod may include drying the second mixture for a period of 10-14 hoursat a temperature range of about 25° C.-37° C. In yet another embodiment,the method may include drying the second mixture for a period of 12hours at a temperature range of about 28° C.-34° C. In certainembodiments, the drying of the second mixture may be accomplished byputting the second mixture in an oven for about 24 hours at 60° C.

The surface modified electrode used in the biosensor allows forultrasensitive detection of biological choline in a non-enzymaticenvironment with higher selectivity, sensitivity, good reliability witha short response time.

EXAMPLES

The disclosure will now be illustrated with examples, which is intendedto illustrate the working of disclosure and not intended to takerestrictively to imply any limitations on the scope of the presentdisclosure.

Example 1: Process of Preparation of Thallium Oxide Carbon NanotubesCarbon Nanocomposites (Tl₂O₃·CNT NCs)

Tl(NO₃)₃, CNT, and NaOH are used as reacting agents in the preparationof Tl₂O₃·CNT NCs by using a simple wet-chemical procedure (WCP) [M. M.Hussain, M. M. Rahman, A. M. Asiri. M. R. Awual. RSC, Non-enzymaticsimultaneous detection of Lglutamic acid and uric acid using mesoporousCo₃O₄ nanosheets, Adv. 6 (2016) 80511-80521, M. M. Hussain, M. M.Rahman, A. M. Asiri. J. Environ, Ultrasensitive and selective4-aminophenol chemical sensor development based on nickel oxidenanoparticles decorated carbon nanotube nanocomposites for greenenvironment, Sci. 53 (2017) 27-38]. WCP is an established solid-statemethod, generally used in the preparation of un-doped or dopednanocomposites. According to this procedure, Tl(NO₃)₃ was dissolvedunder non-stop stirring with distilled water (100.0 mL) in a conicalflask (250.0 mL) and the pH of the resultant solution was maintainedabove 10.0 by addition of NaOH. After 6.0 hours of constant stirring ona hot plate at 90.0° C., the flask was washed methodically withdistilled water and acetone, and kept for drying in the open air forabout 12 hours at room temperature to obtain the Tl₂O₃·CNT NCs. Thalliumoxide nanoparticles (Tl₂O₃ NPs) were also prepared in the same way. Theprepared slurry of Tl₂O₃ NPs and Tl₂O₃·CNT NCs were dried in the oven ata temperature of 60.0° C. for 24.0 h, followed by grinding intoparticles and powders. The particles and powders were dried again in theoven at a temperature of 60.0° C. for 24.0 hours for use inelectrochemical experimentation and applications. A possible mechanismregarding the preparation of Tl₂O₃·CNT NCs is presented in the followingequations (i)-(iv)}.

NaOH→Na⁺+OH⁻  (i)

Tl(NO₃)₃→Tl³⁺+3NO₃ ⁻  (ii)

Na⁺+3OH⁻+Tl³⁺+NO₃ ⁻→Tl(OH)_(3(aq))+NaNO_(3(s))  (iii)

2Tl(OH)_(3(aq))+CNT(Disperssed)→Tl₂O₃·CN_((s)) ⁻+3H₂O  (iv)

Example 2: Preparation of Glassy Carbon Electrode (GCE) with theTl₂O₃·CNT NCs

According to the procedures mentioned by M. M. Hussain and hisco-workers, the required amounts of the glassy carbon electrode (GCE)were prepared and modified as provided in the literature N. M. Hussain,M. M. Rahman, M. N. Arshad, A. M. Asiri., Trivalent Y3+ ionic sensordevelopment based on(E)-Methyl-N′-nitrobenzylidene-benzenesulfonohydrazide (MNBBSH)derivatives modified with nafion matrix, Sci. Rep. 7 (2017) 5832, M. M.Hussain, M. M. Rahman, M. N. Arshad, A. M. Asiri., ElectrochemicalDetection of Ni2+ Ions Using Synthesized(E)-N′-Chlorobenzylidene-4-methylbenzenesulfonohydrazide DerivativesModified with a Nafion Matrix, ChemistrySelect 2 (2017) 7455-7464, M. M.Hussain, A. M. Asiri, M. N. Arshad, M. M., Synthesis, characterization,and crystal structure of(E)-N′-(4-Bromobenzylidene)-benzenesulfonohydrazide and its applicationas a sensor of chromium ion detection from environmental samples, RahmanJ Molecular Structure 1207 (2020) 127810]. A phosphate buffer adapted toa range of pH values, from light acidic to basic phases such as pH=5.7,6.5, 7.0, 7.5, and 8.0, was prepared by dissolving NaH₂PO₄ in distilledwater. Further, the GCE is washed with distilled water and then acetonesystematically and was positioned to dry in the open air for about 1.30hours. Tl₂O₃ NPs, Tl₂O₃·CNT NCs and CNT were mixed with ethanol (EtOH)to prepare a slurry. The slurry was applied on the dried upper surfaceof the GCE. The GCE was further covered, and was placed in the open airto dry for about 1.30 hours. Polymer conducting matrix such as nafion(NFN) was added to the covered GCE in a dropwise manner and kept in anopen environment for about 2.0 hours so as to synchronize the coatingdevelopment. Platinum wire was used as a counter-electrode, and thesurface modified GCE was used as a working electrode to document theelectrical responses associated with identification and detection of thebiological molecule.

Materials and Methods

Thallium nitrate [Tl(NO₃)₃], carbon nanotubes (CNT), acetylcholine,ascorbic acid, cholesterol, choline, dopamine, folic acid, L-glutamicacid, L-glutathione, L-tyrosine, and uric acid, ethanol, nafion, andNaOH were received from the Sigma-Aldrich company (KSA). UV-Visible andFourier transform-infrared (FTIR) spectra of the prepared Tl₂O₃ NPs andTl₂O₃·CNT NCs were recorded respectively on a Thermo scientific 300 kUV-Visible spectrophotometer and NICOLET iS50 FTIR spectrometer(Madison, USA). X-ray diffraction (XRD) experimentation was performedunder ambient environment to determine the crystalline nature of Tl₂O₃NPs and Tl₂O₃·CNT NCs. Field emission scanning electron microscope(FESEM) (JSM-7600F, JEOL, and Japan) attached with energy-dispersiveX-ray spectroscopy (EDS) was used to analyse the electrochemicalcriteria (arrangement, elemental analysis, morphology, and particlesize) of the Tl₂O₃ NPs and Tl₂O₃·CNT NCs had been recorded. Bindingenergy between Cu and O was determined by means of X-ray photoelectronspectroscopy (XPS) experimentation on a thermo scientific A1 K-α1 1066spectrometer having an excitation radiation resource (Beam spotsize=300.0 μm, pressure=10⁻⁸ torr, and pass energy=200.0 eV).Current-voltage experimentation was performed based on an electrometer(Keithley, USA) at a selective point in order to measure desiredsensitive and selective biological molecule by using the sensor of thepresent disclosure (Tl₂O₃·CNT NCs/GCE/NFN).

Results and Discussion Examination of Optical Characteristics

FIG. 2A and FIG. 2B shows UV examination results and bandgap energiesexamination results of Tl₂O₃ NPs. Bandgap energy and spectra of thethallium oxide may be achieved based on the UV-Visible spectroscopyprinciple due to the adsorption of radiant force throughout the movementof the external electrons of the atom to the higher energy phase. FIG.2C and FIG. 2D shows UV examination results and bandgap energiesexamination results of Tl₂O₃·CNT NCs. Wide-ranging absorption UV-visiblecurves of Tl₂O₃ NPs and Tl₂O₃·CNT NCs were achieved at 228.5 nm and230.0 nm which was recorded in the range of 200˜800 nm (as shown in FIG.2A and FIG. 2C). Based on the direct bandgap rule (Tauc's equation, v),theoretical band gap energies (BGE) of the Tl₂O₃ NPs (5.43 eV) andTl₂O₃·CNT NCs (5.39) was achieved. After that hv vs (αhv)2 are plottedand extended to the x-axis (Equations vi-viii) to determine practicalBGE of the Tl₂O₃ NPs (2.60 eV) and Tl₂O₃·CNT NCs (2.80 eV) (as shown inFIG. 2B and FIG. 2D). Here, A=constant related to the effective mass ofthe electrons, h=Plank's constant, v=Frequency, α=absorptioncoefficient, and r=0.5.

FIG. 3A shows Fourier transfer infrared spectroscopy (FTIR) studies ofCNT, Tl₂O₃ NPs, and Tl₂O₃·CNT NCs. FT-IR assessment was performed in theregion of 4000-400 cm⁻¹ under a standard environment to recognize thefunctional character of the Tl₂O₃ NPs and Tl₂O₃·CNT NCs. Reported peaksin 1806, 1315, 1095, 977, and 610 cm⁻¹ were assigned to the presence of—C≡C—, C—H, C—H, ═Tl—O—Tl═, and —Tl═O in the nanocomposite (As shown inFIG. 3A). The assigned peaks at 1095 cm⁻¹ and 610 cm⁻¹ signified theunderstanding of the metal-oxide bond (—Tl═O) confirming the formationof the Tl₂O₃·CNT NCs.

FIG. 3B shows X-Ray Diffraction (XRD) studies of CNT, Tl₂O₃ NPs, andTl₂O₃·CNT NCs. XRD examination was conducted in the range of 2θ=10°-80°to understand the crystalline nature of the Tl₂O₃·CNT NCs. Impendingpeaks intensity with signal for 20 were obtained at 211°, 222°, 400°,431°, 440°, 611°, and 541°, in accordance with the literature values.These potential peaks are an indication of the crystalline nature andpurity of the nanocomposites. According to the XRD examination, it canbe observed that a good number of crystalline Tl₂O₃·CNT was present inthe prepared Tl₂O₃·CNT NCs. By using the Scherer formula (ix), thecrystallite size of the prepared Tl₂O₃ NPs and Tl₂O₃·CNT NCs were foundas 341.32 nm and 34.34 nm respectively.

Referring to FIG. 4A, FIG. 4B, and FIG. 4C, shows field emissionscanning electron microscope (FESEM) images of CNT, Tl₂O₃ NPs, andTl₂O₃·CNT NCs. FESEM was used to examine the morphological properties ofTl₂O₃·CNT NCs. Elemental characteristics of the arranged Tl₂O₃·CNT NCswere examined using FESEM equipped with X-ray energy dispersivespectroscopy (XEDS). Characteristic FESEM images of the CNT, Tl₂O₃ NPs,and Tl₂O₃·CNT NCs were recorded from low to high exaggerated range(diameter of Tl₂O₃·CNT was found to be in the range of 8.0 nanometers to15.0 nanometers, specifically 10.0 nanometers). FIG. 3D shows elementalanalysis of Tl₂O₃·CNT NCs. Based on the XEDS experimentation, it wasobserved that carbon (C), thallium (Tl), and oxygen (O) were found inthe prepared Tl₂O₃·CNT NCs. These nanocomposites consist of carbon(32.03%), oxygen (29.41%), and thallium (38.57%) in weight. Noadditional peaks suggesting impurities were observed in the analysis,confirming that that the nanocomposites formed were only composed ofcarbon, thallium, and oxygen.

FIG. 5A shows comparative binding energy examination of CNT, Tl₂O₃ NPs,Tl₂O₃·CNT NCs. The binding energy examination is calculated based onX-ray photoelectron spectroscopy (XPS) used to discover the materialenvironment of the fundamentals found in the prepared Tl₂O₃·CNT NCs.Electron number and kinetic energy of a material may be projectedthroughout XPS examination in which the X-ray may get altered by thepresence of nanocomposites. Chemical and electronic nature, fundamentalsymphony, and the empirical principle of the elements presented inmaterial might be examined by using this process. Based on the XPSexamination, carbon, thallium, and oxygen were present in the preparedTl₂O₃·CNT NCs (As shown in FIG. 5A). FIG. 5B, FIG. 5C and FIG. 5D referto the binding energy examination of Tl³⁺, C1s, and O1s, respectively.Carbon, oxygen, and spin-orbit thallium were assigned in the major peaksat C1s {285.2 (CNT) and 288.8 (NCs)}, O1s {531.0 (NPs) and 535.0 (NCs)},and Tl³+{118.4, 122.8 (NPs) and 122.6, 126.8 (NCs)}, eV in that orderindicating that Carbon (C), oxygen (O²⁻), and thallium (Tl³⁺) werepresent in the prepared Tl₂O₃·CNT NCs.

FIG. 6 is a flowchart depicting a method for fabrication of Tl₂O₃·CNTNCs on glassy carbon electrode with a polymer matrix (nafion), aproposed electrochemical mechanism of choline at surface modifiedelectrode of the present disclosure, and also depicts outcomes of I-Vexperimental results. Development of the biosensor based on modifiedTl₂O₃·CNT NCs is the preliminary stage in the biological science arena.Tl₂O₃·CNT NCs fabricated GCE were experimented with in phosphate bufferfor the sensitive identification and detection of the biologicalmolecule, like choline. Electrochemical response of the sensor wasdynamically altered due to adsorption of choline during the I-V process.An expected mechanism regarding electrochemical identification anddetection of biomolecule based on the two-electrode system is shown inFIG. 6 . Choline was converted into betaine and hydrogen peroxide inpresence of water and oxygen. Further, hydrogen peroxide was convertedinto proton and oxygen by releasing four electrons. This change inchemical information (loss of electrons) caused by choline on contactwith at least a portion of the working electrode causes the sensor totransduce the change in chemical information associated with choline toan electrical signal. Further, real electrical responses of choline areinvestigated by simple and reliable I-V technique with electrode of thepresent disclosure, which is presented in FIG. 6 . A significantamplification in the current response with applied potential isnoticeably confirmed. Choline in presence of the Tl₂O₃·CNT NCsfabricated GCE sesnor releases 2 electrons to the reaction system, whichimproved and enhanced the current responses against potential during theI-V measurement at room conditions.

pH is an important factor affecting the performance of surface modifiedelectrode. The effect of pH of choline on the sensing ability of thesurface modified electrode was further evaluated, and the results ofthis experiment are presented in FIG. 7A. The surface modifiedelectrodes were examined in phosphate buffer arrangement from loweracidic to little basic condition (pH=5.7-8.0). The pH was adjusted usingchemicals known in the art. All other process parameters were keptidentical while performing the experiment. The current response atdifferent pH was noted. From FIG. 7A, it can be observed that althoughthe electrochemical sensor is effective in detection of choline at awide range of pH values, best results were observed at a slightlyalkaline or neutral pH values. A higher electrical response was recordedat pH=7.5 with the sensor of the present disclosure. From these findingsit is evident that the electrode exhibits superior sensitivity withenhanced current response at pH 7.5, because of the higher rate ofelectron transfer at pH 7.5.

Further, the current response of the surface modified electrode wascompared to that of a bare/uncoated electrode (bare GCE), and GCE coatedwith nafion. The results of this study are presented in FIG. 7B. Adifference in the electrical signal is observed for Tl₂O₃·CNT NCscovered GCE compared to the bare electrode (GCE and GCE including 5%nafion). The results suggest that the Tl₂O₃·CNT NCs.

One of the most essential and desirable features of an electrochemicalsensor lies in its ability to distinguish the analyte of interest, evenat very low concentrations, from interfering chemicals. Because of theability to distinguish interfering agents from choline with very closeelectrochemical behavior, the interference study is one of the importantmethods of analytical chemistry. In other words, the electrochemicalsensor ought to be selective and sensitive. To assess the selectivity ofthe electrode towards choline, 25.0 μL of 1.0 μM choline in (10.0 mL,pH=7.5, and 100.0 mM) phosphate buffer was taken. Nine other interferingchemicals, maintained at the same concentration, were added to a samplecontaining choline. The interfering chemicals are acetylcholine,ascorbic acid, cholesterol, dopamine, folic acid, 1-glutamic acid,1-glutathione, 1-tyrosine, and uric acid. The results of this study arepresented in FIG. 7C. From the FIG. 7C, it can be observed that althoughthe selectivity towards glutamic acid and ascorbic acid was found to bemoderate, best amperometric response was observed, given all otherreaction conditions kept constant, was observed with choline at appliedpotential range of 0.1-1.5 V.

FIG. 7D shows an electrical response of Tl₂O₃·CNT NCs modified electrodein the absence and presence of choline. A control experiment in theabsence and presence of choline (concentration=1.0 μM and amount 25.0μL) was conducted in the buffer phase (10.0 mL, pH=7.5, and 100.0 mM) toobserve the electrical response towards the biomolecule. Tl₂O₃·CNT NCsmodified electrode showed immense response towards choline compared toother fabricated electrodes such as phosphate buffer (PB), bare GCE, andGCE with nafion.

FIG. 8A is an I-V graph depicting the effect of concentration of cholineon current change with the surface modified electrode. Electricalsignals with a concentration range of choline of about 100.0 pM˜100.0 mMwere tested against the proposed sensor (Tl₂O₃·CNT NCs/GCE/NFN) under acustomary arrangement to identify the alteration of responses that areused to recognize the biomolecules. It was recorded that electricalresponses had been augmented on a usual array from low to highconcentration of the choline (SD=0.59, RSD=10.13% at +0.5 V, and n=10).FIG. 8B shows a calibration plot obtained from FIG. 8A vs choline.Calibration bend was plotted at +1.1 V from choline concentration (100.0pM 100.0 mM), observed linear (R2=0.9884), SD=10.08, RSD=32.63%, n=10,and error bar=10.0%. Analytical parameters of the sensor for examplesensitivity (104.68 μAμM-1 cm²), LOD down to 9.14 pM, and LOQ (30.47 μM)were found from calibration bend by means of the equations (x-xii).

$\begin{matrix}{{Sensitivity} = \frac{m}{A}} & (x)\end{matrix}$ $\begin{matrix}{{LOD} = \frac{\left( {3 \times SD} \right)}{m}} & ({xi})\end{matrix}$ $\begin{matrix}{{LOQ} = \frac{\left( {10 \times SD} \right)}{m}} & ({xii})\end{matrix}$

Here, m=slope of the calibration bend (y=3.308 x+29.23), A=dynamicsurface arena of furnished GCE (perimeter=0.0316 cm²), SD=standarddeviation (10.08) of choline concentration in the calibrated impending(+1.1 V) N. M. Rahman, M. M. Hussain, A. M. Asiri., D-Glucose sensorbased on ZnO V205 NRs by an enzyme-free electrochemical approach, RSCAdv. 9 (2019) 31670-31682, M. M. Hussain, A. M. Asiri, M. N. Arshad, M.M. Rahman, A Thallium Ion Sensor Development Based on the Synthesized(E)-N′-(Methoxybenzylidene)-4-Methylbenzenesulfonohydrazide Derivatives:Environmental Sample Analysis, Chemistry Select 4 (2019) 10543-10549, M.M. Rahman, M. M. Hussain, A. M. Asiri, K. A. Alamry, M. A. Hasnat. Anenzyme free detection of L-Glutamic acid using deposited CuO·GdOnanospikes on a flat glassy carbon electrode, Surfaces Interfaces 20(2020) 100617].

FIG. 8C shows a linear dynamic range graph with an error limit of 10%.Linear dynamic range (LDR=1.0 mM˜100.0 pM) is obtained from thecalibration bend and observed linear (R2=0.9778) with linear equation,y=3.324 x+29.25. FIG. 8D shows a response time of choline towards thesensor. Response time of choline (concentration=1.0 μM and amount=25.0μL) towards the sensor (Tl₂O₃·CNT NCs/GCE/NFN) were examined anddetermined in buffer phase (10.0 mL, pH=7.5, and 100.0 mM) and in just4.0 secs.

FIG. 9A is a plot comparing the effect of current change with theTl₂O₃·CNT NCs coated electrode sensor with that of bare electrode sensorin responses to choline. The sensors used to perform this study are GCEmodified with CNTs in presence of nafion; GCE modified with Tl₂O₃ inpresence of nafion; and GCE modified with Tl₂O₃·CNT in presence ofnafion. To understand the electrochemical response of each of thesesensors towards choline, 25.0 μL of 1.0 μM choline in buffer phase (10.0mL, pH=7.5, and 100.0 mM) was taken. It was observed that Tl₂O₃·CNTNCs/GCE/NFN sensor appeared to have a major electrical response comparedto other sensors for example CNT/GCE/NFN and Tl₂O₃ NPs/GCE/NFN. FIG. 8Bshows bar diagram depicting the effect of current change with theTl₂O₃·CNT NCs coated electrode sensor with that of bare electrode sensorin response to choline at +1.2 V with error bar 10.0%. A comparison ofcholine detection with different sensors (prior art) is presented inTable 1.

TABLE 1 Determination of choline using different modified sensorsSensitivity WP (μA mM⁻¹ LOD LDR RT LTS Sensors (mV) cm⁻²) (μM) (μM) (s)(%) AChE-ChO/c-MWCNT 200 — 0.01 0.05-200  4.0 50 ZrO2NPs/GCEChOx/Silicate/MWCNTs/Pt 160 333.0 0.1  5-100 8 91.6, 75.7 ChOx/MnO₂/GC450 24.1 —  10-2100 — 90, 80 PVA/Au nanorods/ChOx/Pt 400 7.2 10  20-40020 80 PDDA/ChOx/Au/MWCNTs 350 186 0.3  1-500 7 82.5(PDDA/ChOx)₃/MnO₂/SPE 480 103 0.13 0.13-100  10 75PDDA/ChOx/ZnO/MWCNTs/PG 400 178 0.3  1-800 13 94.6 Tl₂O₃ · CNTNCs/GCE/NFN 0-1500 104.68 9.14 pM  1.0~100.0 4.0 68, 94 (μAμM⁻¹ (mM~pM)cm⁻²) WP: working potential, LOD: Limit of detection, LDR: Lineardynamic range, RT: response times, LTS: Long term stability, SPE:Screen-printed carbon electrodes.

FIG. 10A and FIG. 10B shows reproducibility and repeatability responseof Tl₂O₃·CNT NCs coated electrode for choline sensing. The effectivenessof the sensor (Tl₂O₃·CNT NCs/GCE/NFN) was evaluated to find out themethodological efficacy such as reproducibility and repeatability. Forthis purpose, a progression of seven subsequent measures 25.0 μL of 1.0μM choline in (10.0 mL, pH=7.5, and 100.0 mM) phosphate buffer wasexperimented with the sensor of the present disclosure. A goodreproducible response was found (RP=68.0%, SD=9.84, RSD=42.92%, and n=7)at the calibrated potential (+1.1 V) (As shown in FIG. 10A).Electrochemical response of the sensor was further analyzed with theintention of expanded storage predisposition. A succession of storageability of the projected sensor was examined by using similar ornamentedelectrode with 25.0 μL of 1.0 μM choline in (10.0 mL, pH=7.5, and 100.0mM) phosphate buffer, and the repeatability was found 94.0% (SD=1.79,RSD=51.9%, and n=7) (as shown in FIG. 10B). It was recorded that theproposed sensor (Tl₂O₃·CNT NCs/GCE/NFN) may be used even after severaldays of analyzing N. M. Hussain, A. M. Asiri, M. M. Rahman, Anon-enzymatic electrochemical approach for 1-lactic acid sensordevelopment based on CuO MWCNT nanocomposites modified with a Nafionmatrix, New J. Chemistry 44 (2020) 9775-9787, M. M. Hussain, A. M.Asiri, M. M. Rahman, Synthesis, characterization, and physicochemicalstudies of the synthesizeddimethoxy-N′-(phenylsulfonyl)-benzenesulfonohydrazide derivatives andused as a probe for calcium ion capturing: Natural sample analysis, J.Molecular Structure 1214 (2020) 128243, M. M. Rahman, M. M. Hussain, A.M. Asiri., Enzyme-free detection of uric acid using hydrothermallyprepared CuO·Fe2O3 nanocrystals, New J. Chem. 44 (2020) 19581-19590].

FIG. 11A is a plot depicting interference analysis of the Tl₂O₃·CNT NCscoated electrode in the presence of other interfering biomolecules.Interference examination is a good analytical exercise for recognitionof biomolecules having biologically similar characteristics andinterfering activity towards the sensor Tl₂O₃·CNT NCs/GCE/NFN [M. M.Rahman, M. M. Hussain, M. N. Arshad, M. R. Awual A. M. Asiri., Arsenicsensor development based on modification with(E)-N0-(2-nitrobenzylidine)-benzenesulfonohydrazide: a real sampleanalysis, New J. Chem. 43 (2019) 9066-9075, A. M. Asiri, M. M. Hussain,M. N. Arshad, M. M. Rahman, A Ce²⁺ sensor based onnapthalen-1-ylmethylene-benzenesulfonohydrazide (NMBSH) molecules:ecological sample analysis, New J. Chem. 42 (2018) 4465-4473, A. M.Asiri, M. M. Hussain, M. N. Arshad, M. M. Rahman, Sensitive andselective heavy metal ion, Mn²⁺ sensor development based on thesynthesized (E)-N0-chlorobenzylidenebenzenesulfonohydrazide (CBBSH)molecules modified with nafion matrix, J. Indust. Engr. Chem. 63 (2018)312-321, M. M. Hussain, A. M. Asiri, M. M. Rahman, Hg2+ SensorDevelopment Based on (E)-N′-NitrobenzylideneBenzenesulfonohydrazide(NBBSH) Derivatives Fabricated on a Glassy Carbon Electrode with aNafion Matrix, ACS Omega 2 (2017) 420-431]. Ascorbic acid, L-lacticacid, and uric acid were used as interfering biomolecules to determinethe electrochemical responses towards the sensor for choline detection.For this purpose, a fluid sample containing 25.0 μL of 1.0 μM choline inbuffer phase (10.0 mL, pH=7.5, and 100.0 mM) was taken and 25.0 μL of10.0 μM of other interfering biomolecules (ascorbic acid, L-lactic acid,and uric acid) were added to determine the selectivity of the sensortowards choline. The actions of interfering biomolecules and cholinetowards the anticipated sensor were calculated in the calibratedpotential (+1.1 V); it was observed that despite the interferingbiomolecules having a concentration 10-fold greater that theconcentration of choline, the selectivity of the sensor towards cholinewas considered to be 100.0%.

TABLE 2 Examination of interference effect towards the sensor (Tl₂O₃ ·CNT NCs/GCE/NFN) Observed current (μA) IF SD RSD % IBM R1 R2 R3 R4 R5Average (%) (n = 3) (n = 30 CHL 16.76 18.31 16.74 3.53 14.28 13.92 1005.99 43.00 AA 14.48 11.52 12.88 10.60 10.13 11.92 86 1.77 14.88 L-LA11.63 9.42 8.67 8.50 8.15 9.27 67 1.40 15.06 UA 7.62 6.89 6.60 6.45 6.246.76 49 0.54 7.93 IBM: Interfering biomolecules, R: Reading, IE:Interfering effects, SD: Standard deviation, and RSD: Relative standarddeviation.

It was found that Tl₂O₃·CNT NCs/GCE/NFN sensor did not show anysignificant electrochemical response towards the interferingbiomolecules having ten times more concentration than choline. Thesensor may be a good investigative appliance to detect sensitivebiological molecules with good selectivity. FIG. 11B shows a bar diagramdepicting current change response to the interfering biomolecules at+1.3 V with an error limit of 10.0%.

FIG. 12 is an I-V graph determining choline concentration from variousbiological samples with the sensor of the present disclosure. For thispurpose, detailed experimentation was accomplished in order to analyzebiological samples for example human serum (HS), mouse serum (MS), andrabbit serum (RS) with Tl₂O₃·CNT/GCE/NFN sensor for identification ofcholine concentration on the basis of a standard addition technique [M.M. Rahman, M. M. Hussain, M. N. Arshad, A. M. Asiri., The synthesis andapplication of(E)-N′-(benzo[d]dioxol-5-ylmethylene)-4-methyl-benzenesulfonohydrazidefor the detection of carcinogenic lead, RSC Adv. 10 (2020) 5316-5327, M.M. Hussain, A. M. Asiri, M. N. Arshad, M. M. Rahman, A Thallium IonSensor Development Based on the Synthesized(E)-N′-(Methoxybenzylidene)-4-Methylbenzenesulfonohydrazide Derivatives:Environmental Sample Analysis, ChemistrySelect 4 (2019) 10543-10549, M.M. Hussain, A. M. Asiri, M. N. Arshad, M. M. Rahman, Fabrication of aGa3+ sensor probe based on methoxybenzylidenebenzenesulfonohydrazide(MBBSH) by an electrochemical approach, New J. Chem. 42 (2018)1169-1180, M. M. Hussain, A. M. Asiri, M. N. Arshad, M. M. Rahman,Development of selective Co2+ ionic sensor based on various derivativesof benzenesulfonohydrazide (BSH) compound: An electrochemical approach,Chem. Engr. J. 339 (2018) 133-143]. A set amount (˜25.0 μL) of eachbiological sample was examined in phosphate buffer (amount=10.0 mL,pH=7.5, and concentration=100.0 mM) and respective calculation wasperformed at the calibrated potential (+1.1 V) to detect cholineconcentration in HS, MS, and RS. The results establish thatcurrent-voltage procedure might be a good experimentation tool for theanalysis of biomolecules in biological sciences area.

TABLE 3 Biological sample analysis AC, CHL OC, RSD (25 μL, CHL BSA ROC(BSA, μA) FC R SD (%) OE μM) (μA) (25 μL) R1 R2 R3 A (μM) (%) (n = 3) n= 3 1 1.0 35.79 HS-1 41.98 38.89 38.70 39.86 1.11 111 1.84 4.62 2 1.022.35 HS-2 19.61 16.71 15.82 17.38 0.78 78 1.98 11.40 3 1.0 24.59 HS-326.08 27.52 25.85 26.48 1.08 108 0.91 3.42 4 1.0 26.17 MS 24.18 21.1919.24 21.54 0.82 82 2.49 11.55 5 1.0 29.51 RS 21.49 21.10 24.26 22.280.76 74 1.72 7.73 OE: Ornamented electrode, AC: Added concentration,CHL: Choline, OC: Observed current, RSA: Real sample added, ROC:Respective observed current, BSA: Biological sample added, R: Reading,A: Average, FC: Found concentration, RC: Recovery, SD: Standarddeviation, RSD: Relative standard deviation, HS: Human serum, MS: Mouseserum, and RS: Rabbit serum.

INDUSTRIAL APPLICABILITY

The biosensor of the present disclosure offers several advantages overthe prior art for detection of choline. One advantage of the embodimentsaccording to the present disclosure is that the biosensor using thalliumoxide-based nanomaterial electrode shows good reliability,reproducibility, and stability under ambient conditions. The Tl₂O₃·CNTNCs coated electrode shows a better electrical response than theuncoated GEC. Another advantage of the biosensor is that it anon-enzymatic electrochemical method for the detection of biomolecules.Yet another advantage of the embodiments of the present disclosure isthe good detectability, high sensitivity, and high selectivity forcholine compounds which is important for the diagnosis of majordiseases. Enhanced electro-catalytic property in detecting choline,handy nature, good reproducibility, wide LDR, high sensitivity, and lowLOD, makes this biosensor an excellent choice for the detection ofcholine.

It is understood that the examples, embodiments and teachings presentedin this application are described merely for illustrative purposes. Anyvariations or modifications thereof are to be included within the scopeof the present application as discussed.

ACKNOWLEDGMENT

The authors extend their appreciation to the Deputyship for Research &Innovation, Ministry of Education in Saudi Arabia for funding thisresearch work through the project number “2021-042” and King AbdulazizUniversity, DSR, Jeddah, Saudi Arabia.

1-12. (canceled)
 13. A method of preparing a surface modified electrode,the method comprising: mixing carbon nanotubes (CNT's), and at least oneof thallium oxide nanoparticles (Tl₂O₃·NPs), thallium oxide (Tl₂O₃)nanopowders, and thallium oxide carbon nanotube nanocomposites(Tl₂O₃·CNT NCs), in an organic solvent to form a slurry of ananomaterial; disposing the slurry of the nanomaterial on a glassycarbon electrode (GCE) to form a film; and coating a polymer matrix onthe film to obtain the surface modified electrode.
 14. The methodaccording to claim 13 further comprising, contacting the slurry of thenanomaterial with the glassy carbon electrode for a period of 1-3 hoursat a temperature range of 35-40° C.
 15. The method according to claim 13further comprising, coating the polymer matrix on the film for a periodof 2-4 hours at a temperature range of 35-40° C.
 16. The methodaccording to claim 13, wherein the polymer matrix is a sulfonatedtetrafluoroethylene-based fluoropolymer (nafion).
 17. The methodaccording to claim 13, wherein the thallium oxide carbon nanotubenanocomposites (Tl₂O₃·CNT NCs) are prepared by: stirring a salt ofthallium in water in an alkaline medium to form a first mixture; washingthe first mixture to obtain a second mixture; and drying the secondmixture to obtain the thallium oxide carbon nanotube nanocomposites. 18.The method according to claim 17 further comprising, stirring the saltof thallium in water in an alkaline medium for a period of 5-7 hours ata temperature range of 80° C.-100° C.
 19. The method according to claim17 further comprising, drying the second mixture for a period of 10-14hours at a temperature range of 25-37° C.
 20. The method according toclaim 18, wherein the salt of thallium is thallium nitrate Tl(NO₃)₃.